A Synbiotic Mixture Augmented the Efficacy of Doxepin, Venlafaxine

Turk J Pharm Sci 2020;17(3):293-298 DOI: 10.4274/tjps.galenos.2019.94210 ORIGINAL ARTICLE

A Synbiotic Mixture Augmented the Efficacy of Doxepin, Venlafaxine, and Fluvoxamine in a Mouse Model of Depression Sinbiyotik Karışım, Fare Depresyon Modelinde Doksepin, Venlafaksin ve Fluvoksaminin Etkinliğini Artırdı

Azadeh MESRIPOUR1*, Andiya MESHKATI1, Valiollah HAJHASHEMI2

1Isfahan University of Medical Sciences, School of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology and Toxicology, Isfahan, Iran 2Isfahan University of Medical Sciences, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan, Iran

ABSTRACT

Objectives: Currently available antidepressant drugs have notable downsides; in addition to their side effects and slow onset of action their moderate efficacy in some individuals may influence compliance. Previous literature has shown that probiotics may have antidepressant effects. Introducing complementary medicine in order to augment the efficacy of therapeutic doses of antidepressant drugs appears to be very important. Therefore, the effect of adding a synbiotic mixture to drinking water was assessed in a mouse model of depression following the administration of three antidepressant drugs belonging to different classes. Materials and Methods: The marble burying test (MBT) and forced swimming test (FST) were used as animal models of obsessive behavior and despair. The synbiotic mixture was administered to the mice’s drinking water (6.25x106 CFU) for 14 days and the tests were performed 30 min after the injection of the lowest dose of doxepin (1 mg/kg), venlafaxine (15 mg/kg), and fluvoxamine (15 mg/kg) on days 7 and 14. Results: After 7 days of ingestion of the synbiotic mixture, immobility time decreased in the FST for doxepin (92±5.5 s) and venlafaxine (17.3±2.5 s) compared to the control group (drinking water), but fluvoxamine decreased immobility time after 14 days of ingestion of the synbiotic mixture (70±7.5 s). Preadministration of the synbiotic mixture improved the MBT test response for venlafaxine, while it did not change the results for the other two drugs. Conclusion: Adding the synbiotic mixture to drinking water improved the efficacy of discrete antidepressant drugs particularly during the FST. Probiotics could be a useful complementary medicine for drug-resistant depressed individuals. Key words: Probiotic, synbiotic, depression, antidepressant, forced swimming test, complementary medicine ÖZ

Amaç: Günümüzde temin edilebilen antidepresan ilaçların belirgin dezavantajları vardır; yan etkilerine ve yavaş etki etmelerine ek olarak, orta derecedeki etkinlikleri bazı kişilerde hasta uyuncunu etkileyebilir. Daha önceki literatür bilgileri probiyotiklerin antidepresan etkilere sahip olabileceğini göstermiştir. Antidepresan ilaçların terapötik dozlarının etkinliğini artırmak için tamamlayıcı tıbbın etkisinin çok önemli olduğu düşünülmektedir. Burdan hareketle, bu çalışmada içme suyuna sinbiyotik bir karışımın eklenmesinin etkisi, farklı sınıflara ait üç antidepresan ilacın uygulanmasını takiben fare depresyon modelinde değerlendirilmiştir. Gereç ve Yöntemler: Obsesif davranış ve umutsuzluğun hayvan modeli olarak misket gömme testi (MBT) ve zorunlu yüzme testi (FST) kullanılmıştır. Sinbiyotik karışım, farelerin içme suyuna (6,25x106 CFU) 14 gün boyunca uygulanmış ve testler, 7. ve 14. günlerde en düşük dozda doksepin (1 mg/ kg), venlafaksin (15 mg/kg) ve fluvoksamin (15 mg/kg) enjeksiyonundan 30 dakika sonra yapılmıştır. Bulgular: Sinbiyotik karışımın 7 gün süreyle uygulanmasından sonra, doksepin (92±5,5 s) ve venlafaksin (17,3±2,5 s) için FST’de kontrol grubuna (içme suyu) kıyasla hareketsizlik süresi azalmış, ancak fluvoksamin ile, sinbiyotik karışımın 14 gün süreyle uygulanmasından (70±7,5 s) sonra hareketsizlik süresi azalmıştır. Sinbiyotik karışımın önceden uygulanması venlafaksin için MBT test yanıtını geliştirmiş, diğer iki ilacın sonuçlarını değiştirmemiştir. Sonuç: Sinbiyotik karışımın içme suyuna eklenmesi, özellikle FST sırasında ayrı antidepresan ilaçların etkinliğini arttırmıştır. Probiyotikler, ilaca dirençli depresif bireyler için yararlı bir tamamlayıcı olabilir. Anahtar kelimeler: Probiyotik, sinbiyotik, depresyon, antidepresan, zorunlu yüzme testi, tamamlayıcı tıp

*Correspondence: E-mail: [email protected], Phone: +00983137927089 ORCID-ID: orcid.org/0000-0003-3150-5581 Received: 04.02.2019, Accepted: 07.03.2019 ©Turk J Pharm Sci, Published by Galenos Publishing House. 293 294 MESRIPOUR et al. Synbiotic Mixture Augmented Antidepressant Drug Efficacy

INTRODUCTION intestinal microflora such as Lactobacilli and Bifidobacteria also Probiotics are live organisms that when chronically ingested in have neurotropic effects.18 adequate amounts could induce beneficial effects in the host.1 Although drug therapy of depression is usually safe and Single strain probiotics or their combinations are useful for a effective, it is still not ideal because the latency time for variety of diseases, including gastrointestinal (GI) disorders clinical results is quite long (about 3-5 weeks) and there are such as inflammatory bowel disease, diarrhea, Irritable still concerns regarding side effects such as loss of libido Bowel syndrome (IBS), and allergies.2 This is now even being and weight gain. Additionally, there are a significant number extended to some ailments of the central nervous system of patients that do not respond well to antidepressant drugs, (CNS).3,4 Gut bacterial microorganisms have two dominant psychotherapy, and electroconvulsive therapy.19 Thus the aim of genera, Bacteroidetes and Firmicutes, while other types have the present study was to first observe the effect of a synbiotic lower abundance, for instance Proteobacteria, Actinobacteria, (probiotic + prebiotic) mixture on behavior in an animal model and Fusobacteria.5 Evidently, the brain-gut axis is a bidirectional of depression. Since it has been previously shown11-15 that the homeostatic route and so its aberration may cause important probiotics Lactobacilli and Bifidobacteria have antidepressant pathophysiological results.3 The high risk of co-existing effects in animal studies, a manufactured premixed product psychiatric symptoms such as anxiety with GI disorders, for was chosen that comprised these genus and also a prebiotic, instance IBS, provides further evidence of the importance of fructooligosaccharides. Second, in order to observe the effect this axis.6 The brain-gut axis is connected not only by complex of the synbiotic mixture on the efficacy of antidepressant systems including the vagus nerve, endocrine, immune, and drugs from different classes, subthreshold doses of doxepin humoral links but also the gut microbiota in order to maintain [a tricyclic antidepressant (TCA)], venlafaxine [a serotonin- GI stability and to connect cognitive and emotional areas of the norepinephrine reuptake inhibitor (SNRI)), and fluvoxamine (a brain with GI functions.7 selective serotonin reuptake inhibitor (SSRI)] were chosen. Major depressive disorder is the most common mood disorder, affecting 5% of the population each year.8 Several mechanisms MATERIALS AND METHODS are involved in its pathogenesis, such as altered monoaminergic Animals (serotonergic, noradrenergic, and dopaminergic) and Male Swiss mice weighing 23-26 g were housed at 21±2 ºC glutamatergic systems, increased inflammation, hypothalamic- in a 12-h light/dark cycle with the lights on in the day time 6 pituitary-adrenal (HPA) axis aberrations, and decreased am-6 pm. Tap water or the synbiotic mixture and standard 9 neuroplasticity. Probiotics have shown antidepressant food pellets were available ad libitum. For each experiment effects in animal models of depression. Species of the genus 6 mice were used and they were housed 3 per cage. Tests 10 Lactobacillus are particularly characterized as antidepressants. were performed in the behavior laboratory 24 h after the A probiotic mixture of Lactobacillus strains (L. rhamnosus and L. mice had become acclimatized to the environment. In order to helveticus) ameliorated depression by regulating corticosterone minimize the influence of circadian rhythm, all experiments 11 levels in rat pups. Likewise, chronic ingestion of a L. rhamnosus were performed between 8 am and 1 pm in the pharmacology strain (JB-1) in mice alleviated depressive behavior and caused laboratory. All animal procedures were approved by the Ethics regional alteration in GABA receptor expression and since Committee of Isfahan University of Medical Science and these changes were not observed in vagotomized mice the performed in accordance with the National Institutes of Health 12 vagus nerve was considered critical for the brain-gut axis. Guide for the Care and Use of Laboratory Animals (Ethical no: Bifidobacterium species also induce physiologic changes in IR.MUI.REC.1395.3.864). favor of antidepressants effects in animal studies; for instance, B. infantis ameliorated depressive behavior in the maternal The synbiotic mixture and drug therapy separation model.13 In a systematic review regarding the effects The synbiotic mixture comprised 109 CFU [Lactobacillus casei, L. of probiotics on depression in humans most of the studies found acidophilus, L. rhamnosus, L. bulgaricus, Bifidobacterium breve, positive results on all values of depressive symptoms, but there B. infantis, Streptococcus thermophilus, fructooligosaccharides were wide differences in the strain of probiotic, the dosing, and (a prebiotic); a product of Zist Takhmir, Iran]. The animals had the duration of treatment.14 The possible mechanisms that were free access to the synbiotic mixture solution in drinking water considered for antidepressant effects of B. infantis by use in a rat that was prepared freshly each day in three concentrations: model were reduction of pro-inflammatory cytokines, alteration 6.25, 12.5, and 25x106 CFU (based on pilot studies); the control of tryptophan metabolism, and CNS neurotransmitters.15 The animals had tap water ad libitum. The amounts of the synbiotic combination of Lactobacilli and Bifidobacteria was tested in mixture solution or normal drinking water ingested by the depression following post-myocardial infarction (MI) in rats. animals were measured daily. The therapy continued for 14 Administration of L. helveticus and B. longum together had days and the tests were performed on days 7 and 14. positive effects on post-MI depression through reduction of The antidepressants were first administered intraperitoneally pro-inflammatory cytokines (for instance IL-4) and intestinal alone 30 min before starting the first test. Two doses were permeability restoration.16,17 In addition, prebiotics such as applied for each drug according to previous studies20 and finally oligosaccharides that stimulate the growth of nonpathogenic the lowest effective dose (data not shown) was chosen to be MESRIPOUR et al. Synbiotic Mixture Augmented Antidepressant Drug Efficacy 295

applied following the synbiotic mixture administration. The consuming 12.5x106 CFU synbiotic after 7 days or 14 days selected antidepressants were doxepin 1 mg/kg (Razak, Iran), reduced obsessive behavior in the mice (Figure 1A). The venlafaxine 15 mg/kg (Sigma-Aldrich, India), and fluvoxamine percentage of marbles buried after 30 min on days 7 and 14 of 15 mg/kg (Sigma-Aldrich, India). All the drugs were freshly ingesting the middle concentration of the synbiotic mixture was prepared in normal saline solution and injections were adjusted 25% and 41%, respectively, which was significantly (p<0.05) for a volume of 10 mL/kg mouse body weight. The selected different from the corresponding control group (50% and 58.3% dose of each antidepressant drug was administered on days 7 on days 7 and 14, respectively; p<0.05). The lowest synbiotic and 14 following the synbiotic therapy (6.25x106 CFU), 30 min concentration only reduced MB to a significant amount on day before the tests were performed. 7 compared to the control animals (33.3% vs 50%; p<0.05). However, consuming the highest synbiotic concentration Marble burying test (MBT) showed opposite results on MB behavior, since the animals The first test was the MBT. This is a method used to evaluate buried more marbles measured after 7 (87%, p<0.001 vs control) anxiety behavior and obsessive compulsive behavior. With minor and 14 days of therapy. During the FST the animals that ingested 21 modification to the method presented by Njung’e and Handley , the synbiotic showed antidepressant behavior as presented in the mice were separately placed in plastic cages (42x24x12 cm) Figure 1B. After the mice had ingested the synbiotic mixture for containing 12 opaque glass marbles (1 cm diameter) that were distributed evenly over 5 cm of deep sawdust without food or water for 30 min. The number of marbles at least two-thirds buried (MB) was counted after 30 min.22 Forced swimming test (FST) After performing the MBT each mouse was subjected to the FST. With some modifications23 the mice were forced to swim in a glass beaker (diameter 14 cm) containing 25 ºC water for 6 min. The depth was about 15 cm to prevent the mice from escaping or touching the bottom of the glass beaker with their paws or tail. After 2 min of adaptation the total immobility time was measured by a chronometer in the last 4 min of the trial. Immobility is defined as the time the animal was floating and staying still when no additional activity was observed other than that required to keep the animal’s head above water. Finally the mice were dried carefully and returned to their home cage. Data processing and statistical analysis The results were expressed as group mean ± standard error of the mean. The results were analyzed by One-Way ANOVA, followed by Tukey’s multiple comparison tests and p values less than 0.05 were considered significant. The software programs used for data analysis and making graphs were Excel 2010 and the GraphPad Prism 6. Figure 1. The effect of 3 concentrations of the synbiotic cocktail ingestion RESULTS for 14 days on mouse behavior. A) The MBT the number of marbles at least two-thirds buried after 30 min, and B) the total immobility time during the Daily drinking intake last 4 min in the FST. The tests took place on days 7 and 14; each animal was first subjected to the MBT and then the FST. Number of animals in each According to Table 1, daily measurements of the synbiotic group was 6; the control animals drank tap water. Results are expressed as mixture ingestion showed that approximately a dose of 2.4- group mean ± SEM and analyzed by One-Way ANOVA, followed by Tukey’s 9.2x106 CFU/g body weight of synbiotic was ingested. post-hoc test. * p<0.05, ** p<0.01, and *** p<0.001 compared with the control group Effect of the synbiotic mixture on the MBT and FST MBT: Marble burring test, FST: Forced swimming test, SEM: Standard error of the The number of marbles buried during the MBT showed that mean

Table 1. Daily liquid intake Synbiotic mixture CFU 6.25x106 12.5x106 25x106 0 (Tap water) Daily intake mL/g body weight 0.38 0.37 0.37 0.39 Daily dose CFU/g body weight 2.375x106 4.625x106 9.25x106 0

The synbiotic mixture comprised Lactobacillus casei, L. acidophilus, L. rhamnosus, L. bulgaricus, Bifidobacterium breve, B. longum, Streptococcus thermophilus, fructooligosaccharides (a prebiotic) 296 MESRIPOUR et al. Synbiotic Mixture Augmented Antidepressant Drug Efficacy

a week the immobility time measured during the FST decreased synbiotic mixture did not have any considerable effect on the in a dose-dependent manner since the highest mixture MB behavior of animals injected with doxepin or fluvoxamine. concentration (25x106 CFU) caused the lowest immobility time On the other hand, drinking the synbiotic mixture increased the (68.1±2.5 s vs 142.3±4.3 s, p<0.001). After 14 days of synbiotic antidepressant effects of the lowest dose of each of the drugs ingestion, the animals dose-dependently showed a shorter (Figure 2B). The immobility time decreased dramatically in the immobility time compared with the control animals (139.6±5.4 animals that had drunk the synbiotic mixture either for 7 days s); the animals that had ingested the highest concentration of (17.3±2.5 s) or for 14 days (60±16 s) prior to the administration the synbiotic mixture had the shortest immobility time (58.3±15 of venlafaxine (p<0.001, vs the venlafaxine alone group 146±1 s, p<0.001). s). The results for synbiotic drinking prior to doxepin injection followed the same trend; after 7 days the immobility time Effect of the synbiotic mixture pretreatment on mouse response was 92±5.5 s and after 14 days it was 70±7.4 s, which was to antidepressants during the MBT and FST decreased significantly compared to the doxepin alone group, The lowest concentration of the synbiotic mixture (6.25x106 100±19 s (p<0.001). The synbiotic effect on fluvoxamine was CFU) was chosen to observe the animals’ behavior during the slightly different as only after 14 days immobility time decreased MBT and FST on days 7 and 14 after injection of the lowest dose significantly compared to the drug administered alone (70±7.5 of each antidepressant drug (Figure 2). The antidepressant s vs 100±9 s, p<0.001). drugs all significantly reduced the number of MB compared to the control group (Figure 2A, p<0.05). Synbiotic ingestion DISCUSSION significantly influenced the effects of venlafaxine on MB Our results showed that the synbiotic mixture could mitigate behavior since the animals significantly buried fewer marbles the immobility time in the FST, which denotes its possible compared to those that had drunk water (percentage of MB antidepressant effects. Interestingly, only after a week of decreased from 25% to 7%). However, consumption of the ingesting the synbiotic mixture did response to the lowest dose of the antidepressants increase dramatically. However, the MB behavior showed variable results. The MB behavior appears to be just a form of digging, but the behavior in mice has been used as a model of anxiety disorders including obsessive compulsive disorder (OCD).24 This method is also useful for evaluating the mechanisms of action of drugs.25 Venlafaxine (SNRI), doxepin (TCA), and fluvoxamine (SSRI) in our set of experiment clearly reduced the number of marbles buried, which was parallel with previous literature.22,25 The FST is a reliable tool for drug discovery in industrial settings where high quantity screening of new compounds is necessary, as well as in research regarding complementary medicine.23 By leaving the mouse in the water container it gradually loses hope to escape the stressful environment, and thus the immobility time reflects a measure of “behavioral despair”. In the same trend as previous results, all antidepressant drugs of different classes that were tested in our study presented antidepressant effects in mice by reducing the immobility time.22 The daily preparation of the synbiotic mixture in the mice’s drinking water was based on previous literature that B. infantis was administered by dissolving a powder containing 1010 bacterial cells in 100 mL of the rats’ drinking water every morning.26 There is also evidence that dead probiotic bacteria or just Figure 2. The effect of drinking the synbiotic mixture and the lowest dose integral components of the bacterial cell such as peptidoglycan of antidepressants on mouse behavior. A) The MBT the number of marbles fragments or DNA would also be effective.27 Therefore, although at least two-thirds buried after 30 min are presented, and B) the total survivability of the mixture was not assessed before the new immobility time during the last 4 min in the FST. Number of animals in each of the groups was 6; animals ingested the synbiotic mixture (SYN; 6.25x106) batch the next morning the research showed that the content for 14 days; the control group ingested tap water. The drugs were injected remained effective. The synbiotic mixture dose-dependently IP 30 min before testing; each animal was first subjected to the MBT and reduced the immobility time during the FST, which obviously then the FST. Results are expressed as group mean ± SEM and analyzed denotes its antidepressant-like effect in mice. However, a by One-Way ANOVA followed by Tukey’s post-hoc test. ** p<0.01 and *** dose-dependent effect on obsessive behavior was not observed p<0.001 compared with the probiotic free group. # p<0.01 compared with the corresponding control groups (the plane bars) during the MBT; although lower doses of the synbiotic mixture IP: Intraperitoneally, MBT: Marble burying test, FST: Forced swimming test, SYN: reduced the MB behavior, this was not observed with the higher Synbiotic, SEM: Standard error of the mean dose. A high dose of the synbiotic mixture had opposite effects MESRIPOUR et al. Synbiotic Mixture Augmented Antidepressant Drug Efficacy 297

on the MBT. The different effects of the synbiotic mixture on days of the synbiotic drink it had a synergistic antidepressant- the FST and MBT could be because of the different mechanisms like effect on venlafaxine and doxepin, and it increased involved in each test. Earlier research has proven that although fluvoxamine efficacy after 14 days. Previous results proved the MBT and FST are invaluable predictive tests of OCD that fluoxetine and escitalopram have antimicrobial activity and antidepressant action, each assay appears to engage in vitro, and psychotropic medications differentially influence completely distinct neurochemical systems.28 While the FST is the composition of gut microbiota in vivo.32 Therefore, adding more susceptible to compounds that are effective by altering probiotics may help to improve the gut microbial composition in activity in the noradrenergic system, the MBT mostly depends patient receiving antidepressant therapies. on modulation of the serotonergic system.28 The downside of our research was that we did not measure the monoamine CONCLUSION 29,30 neurotransmitters, but this has been proven earlier. Reports Since extrapolating animal results to humans must be done with have shown that chronic gavage of in mice L. plantarum caution, further clinical research is warranted regarding adding reduced stress-induced depression-like behaviors, normalized synbiotics to drug-resistant patients and adding synbiotics in the HPA axis and immune systems, and modulated the changes order to reduce antidepressant drug dosage and side effects in the dopamine and serotonin system in the prefrontal cortex.29 mainly when SNRI or TCA are administered. Additionally, probiotics changed behavior and the CNS function 30 in naïve adult animals. The consumption of a fermented milk ACKNOWLEDGEMENT product containing B. aimalis, S. thermophilus, L. bulgaricus, and L. lactis in healthy women without psychiatric symptoms as This work was supported by the School of Pharmacy and observed by functional magnetic resonance imaging induced Pharmaceutical Sciences Research Council (grant number: robust alterations in the activity of brain regions that control the 395864, 12/05/2016), Isfahan University of Medical Sciences. central processing of emotions and sensations.31 Conflict of Interest: Authors confirm that there is no conflict of Pretreatment with the synbiotic mixture before the administration interest in relation to this article. of venlafaxine (SNRI) had a synergistic effect on the MBT, but this synergistic effect was not observed with doxepin or fluvoxamine. Previous studies on rat models of depression have REFERENCES reported that the ingestion of probiotics has a role in restoring 1. Sherman PM, Ossa JC, Johnson-Henry K. 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